To date, about 200 zoonotic diseases (bartonelosis, leptospirosis, Lyme borreliosis, etc.), which affect humans have been described. In third world countries, they represent one of the main causes of death and entail substantial economic loss. Coexistence with animals, lack of sanitary infrastructure and low cultural level continue to be the main allies of these diseases.
Certain types of zoonosis are now thriving in industrialized countries as a consequence of population increases in urban and periurban areas, and increased movement of animals across international borders, which entails the risk of introducing exotic diseases into the environment.
These circumstances, coupled with the frequent findings of arthropods infected by more than one of the pathogens included in the present invention, increase the possibility of more than one of the bacterial species included in the present invention being transmitted in a single sting.
As a result, hospitalizations due to medical profiles produced by human contact with animals or different classes of arthropods, such as mosquitoes, ticks, fleas, lice, mites, etc., which act as vectors or pathogen reservoirs, is becoming increasingly common. Said medical profiles, due to their high degree of similarity, do not allow a fast and reliable identification of the pathogenous agent, so that specific and fast treatment is not possible and is occasionally administered too late. This undoubtedly justifies the need for a comprehensive detection method.
The diagnostic methods currently available are limited to detecting antibodies which, in general, is retrospective and of little use to treating patients in acute-phase states. Culture is not considered a diagnostic method, due both to its technological complexity, which excludes it from regular practice in hospital microbiology laboratories, and to the need for P3 facilities.
Molecular diagnosis by genome amplification by means of PCR represents a diagnostic option of great value. However, clinical samples of sufficient quantity for pathogen testing or the methodology required to carry out different tests are not always available.
A paper has recently been published (Blaskovic D. et al. 2005. Oligo-based detection of tick-borne bacteria. FEMS Microbiology Letters 243:273-8) which describes a method for the detection of 5 out of 6 pathogens proposed by the present invention. Said method is based on ribosomal DNA analysis and uses universal primers, which amplify the genetic material of both target and non-target bacteria, due to which its sensitivity is substantially reduced.
Other methods, such as those described by U.S. Pat. Nos. 6,300,072 and 6,518,020, are capable of detecting and identifying bacteria of the genus Bartonella, by using the same DNA region (intergenic space 16S-23S). However, the number of species within this genus has increased substantially since said patents were filed and their approximation, which consists of discriminating between species according to the size of the amplicon obtained during PCR, is not useful for certain known species within the same genus which are similar in size to the amplified fragment.
While the method provided by the present invention also proposes using intergenic region 16S-23S for the detection of species belonging to the genus Bartonella, improvements have been introduced with respect to the previously described procedures, as it is capable of detecting a much wider range of species within the same and other genera, using completely new probes and primers with maximum sensitivity levels.
For the detection of Coxiella burnetii, the present invention uses the same primers and DNA region (insertion sequence IS1111) as the previously described methods. Said detection has been improved by combining it with another series of completely new tests aimed at identifying other bacterial species, which can be transmitted by the same vectors and also provide a new hybridization probe for the detection of Coxiella burnetii. 